Function of L-Type Ca2+ Channels in B-Lymphocytes

描述(申请人提供)：钙(Ca~(2+))作为一种通用信使，通过调节细胞内水平，使细胞能够有效地对细胞外信号作出反应，在生物学中发挥着核心作用。因此，细胞膜对钙离子的通透性受到称为离子通道的膜蛋白的仔细调节。建立有效的免疫反应还需要延长淋巴细胞等免疫细胞内钙离子的升高时间。B淋巴细胞是适应性免疫系统中负责产生抗体的细胞。B淋巴细胞表达的离子通道的多样性比最初预期的要大，包括L类型的Cav通道变体等分子，这些分子最初被认为是针对所谓的可兴奋细胞，如心脏或神经细胞。虽然在B细胞中存在这些L类通道已被证实，但它们的功能仍有待阐明。这项建议的目的是建立遗传模型系统，使我们能够研究L类通道家族的个体成员在B细胞环境中的作用。这包括缺乏L通道的B细胞系的产生和功能鉴定，随后可以用野生型和突变型通道来补充。我们还计划通过在现有的小鼠模型中研究B细胞限制性基因组缺失L型通道的影响来分析这些分子在体内的功能。由于免疫反应是身体多个位置的多种细胞类型的非常复杂的相互作用，这个体内模型代表了对细胞系的补充方法，将使我们能够在整个生物体的背景下研究L类型通道在B细胞发育和激活中的潜在作用。公共卫生相关声明：L型钙通道是包括维拉帕米和地尔硫卓在内的一类药物的重要治疗靶点，这两种药物通常用于治疗高血压等疾病。体外研究表明，这些药物也抑制免疫细胞的钙反应，尽管浓度高于治疗用的这些药物。这表明这些通道的免疫特异性变体对这些化合物不那么敏感。因此，开发免疫调节药物的一个有希望的途径是设计这些已知通道抑制剂的修饰版本，从而特异性地结合和抑制L类型通道的免疫系统版本。为了达到这个目的，必须确定这些通道在免疫环境中表达的功能变体，以及它们在免疫反应激活过程中的确切作用。此外，这些发现也可能有利于患有影响L型经络功能的罕见复杂遗传疾病的年轻患者，他们的症状与免疫缺陷一致。这一建议将有助于通过研究这些通道在B淋巴细胞中的作用来回答这些重要的问题。

DESCRIPTION (provided by applicant): Calcium (Ca2+) plays a central role in Biology as a universal messenger by allowing cells to efficiently respond to extracellular signals via modulation of its intracellular level. The permeability of cellular membranes for Ca2+ ions is therefore carefully regulated by membrane proteins called ion channels. The mounting of an efficient immune response also requires an extended period of intracellular Ca2+ elevation in immune cells such as lymphocytes. B-lymphocytes are the cells of the adaptive immune system that are responsible for the production of antibodies. The diversity of ion channels expressed in B lymphocytes is greater than originally anticipated, and includes molecules such as L-type CaV-channel variants that were originally assumed to be specific for so-called excitable cells, such as cardiac or neuronal cells. Although the presence of these L-type channels in B-cells is well established, their function remains to be elucidated. The aim of this proposal is to establish genetic model systems allowing us to investigate the role of individual members of the L-type channel family in the B-cell context. This includes the generation and functional characterization of B-cell lines lacking L-type channels that can subsequently be complemented with wildtype and mutant versions of the channels. We also plan to analyze the function of these molecules in vivo by studying the effect of B-cell restricted genomic deletion of L-type channels in an existing mouse model. Because the immune response is a very complex interplay of numerous cell types in multiple locations of the body, this in vivo model represents a complementary approach to the cell lines that will allow us to study the potential role of L-type channels in B-cell development and activation in the context of the complete organism. Public Health Relevance Statement: L-type Ca2+ channels are important therapeutic targets of a class of drugs that include verapamil and diltiazem, which are commonly used to treat conditions such as hypertension in patients. It has been shown in vitro that these drugs also inhibit the Ca2+-response of immune cells, although at higher concentrations than these applied therapeutically. This suggests that immune specific variants of these channels exist that are less sensitive towards these compounds. A promising avenue of immuno-modulatory drug development is therefore to design modified versions of these known channel inhibitors that would specifically bind and inhibit the immune system versions of L-type channels. To this aim, it is essential to identify the functional variants of these channels expressed in the immune context, and their precise role during immune response activation. Furthermore, these findings could also be beneficial to young patients suffering from rare complex genetic disorders affecting L-type channel function, and who are known to show symptoms consistent with immuno-deficiencies. This proposal will contribute to answering these important questions by investigating the role of these channels in B-lymphocytes.